Expression of herbicide tolerance genes in plant plastids

ABSTRACT

Provided are constructs and methods for expressing herbicide tolerance genes in plastids of plant cells. Constructs include the components of a promoter functional in a plant plastid, a DNA sequence which is capable of conferring tolerance in a plant cell to at least one herbicide compound when said DNA sequence is transcribed in plastids of said plant cell and a transcription termination region. Herbicide tolerance is produced by transforming plastids with the constructs of the invention and growing plant cells comprising the transformed plastids under conditions wherein the DNA sequence is transcribed and plant plastids and cells containing the plastids are rendered tolerant to applications of at least one herbicide compound.

This application is a continuation-in-part of application Ser. No.09/113,257 filed Jul. 10, 1998, now Abandoned.

TECHNICAL FIELD

This invention relates to the application of genetic engineeringtechniques to plants. Specifically, the invention relates tocompositions and methods for enhancing expression of proteins in plantplastids.

BACKGROUND

The plastids of higher plants are an attractive target for geneticengineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts,etioplasts, chromoplasts, etc.) are the major biosynthetic centers that,in addition to photosynthesis, are responsible for production ofindustrially important compounds such as amino acids, complexcarbohydrates, fatty acids, and pigments. Plastids are derived from acommon precursor known as a proplastid and thus the plastids present ina given plant species all have the same genetic content. Plant cellscontain 500-10,000 copies of a small 120-160 kilobase circular genome,each molecule of which has a large (approximately 25 kb) invertedrepeat. Thus, it is possible to engineer plant cells to contain up to20,000 copies of a particular gene of interest which potentially canresult in very high levels of foreign gene expression. In addition,plastids of most plants are maternally inherited. Consequently, unlikeheterologous genes expressed in the nucleus, heterologous genesexpressed in plastids are not pollen disseminated, therefore, a traitintroduced into a plant plastid will not be transmitted to wild-typerelatives.

There remains a need for improved regulatory elements for expression ofgenes in a plant plastid. To date, the expression signals used routinelyfor plastid transgene expression derive from endogenous plastid genes.The plastid expression signals are typically derived from promoterregions of highly expressed plastid genes such as the promoter regionsfrom the 16S ribosomal RNA operon (Prrn), psbA gene (PpsbA) or the rbcLgene (PrbcL). The psbA and rbcL genes are highly transcribed, but theirtranslation is controlled by tissue-specific and light-regulated factorswhich limits their usefulness. In the case of Prrn, a synthetic ribosomebinding site (RBS) patterned after the plastid rbcL gene leader has beentypically used to direct translation. However, this Prrn/RBS istranslated inefficiently due to poor ribosome binding.

A totally heterologous expression system has been used to expressplastid genes (U.S. Pat. No. 5,576,198, the entirety of which isincorporated herein by reference). This system is a two componentsystem. The first component is a plastid transgene driven by a T7bacteriophage gene 10 promoter/leader sequence. The second component isa nuclear gene encoding the T7 Polymerase that is targeted to theplastid compartment. The limitation of this system is the need to createnuclear transformed lines that express the T7 Polymerase in preferredways.

Plastids of higher plants present an attractive target for geneticengineering As mentioned above, plastids of higher plants are maternallyinherited. This offers an advantage for genetic engineering of plantsfor tolerance or resistance to natural or chemical conditions, such asherbicide tolerance, as these traits will not be transmitted towild-type relatives. In addition, the high level of foreign geneexpression is attractive for engineered traits such as the production ofpharmaceutically important proteins.

Expression of nucleic acid sequences encoding for enzymes providing forherbicide tolerance as well as pharmaceutical proteins from plantplastid genome offers an attractive alternative to expression from theplant nuclear genome.

SUMMARY OF THE INVENTION

The present invention provides nucleic acid sequences useful inenhancing expression of a wide variety of genes, both eukaryotic andprokaryotic, in plant plastids. Furthermore, plastid expressionconstructs are provided which are useful for genetic engineering ofplant cells and which provide for enhanced expression of the EPSPsynthase proteins or the hGH protein in plant cell plastids. Thetransformed plastids should be metabolically active plastids, and arepreferably maintained at a high copy number in the plant tissue ofinterest, most preferably the chloroplasts found in green plant tissues,such as leaves or cotyledons.

The plastid expression constructs for use in this invention generallyinclude a plastid promoter region capable of providing for enhancedexpression of a DNA sequence, a DNA sequence encoding an EPSPS proteinor hGH, and a transcription termination region capable of terminatingtranscription in a plant plastid.

The plastid promoter region of the present invention is preferablylinked to a ribosome binding site which provides for enhancedtranslation of mRNA transcripts in a plant plastid.

The plastid expression construct of this invention is preferably linkedto a construct having a DNA sequence encoding a selectable marker whichcan be expressed in a plant plastid. Expression of the selectable markerallows the identification of plant cells comprising a plastid expressingthe marker.

In a preferred embodiment, vectors for transfer of the construct into aplant cell include means for inserting the expression and selectionconstructs into the plastid genome. This preferably comprises regions ofhomology to the target plastid genome which flank the constructs.

The constructs of the present invention preferably comprises a promotersequence linked to a ribosome binding site capable of enhancing thetranslation of mRNA transcripts in the plant plastid. The ribosomebinding site is preferably derived from the T7 bacteriophage gene 10leader sequence.

Of particular interest in the present invention is the high level ofexpression of nucleic acid sequences in plant plastids. Of particularinterest is the high level expression of nucleic acid sequences encodingfor enzymes involved in herbicide tolerance and encoding forpharmaceutical proteins.

The constructs of the present invention preferably comprises a DNAsequence encoding for a 5-Enolpyruvylshikimate-3-phosphate synthase(U.S. Pat. No. 5,633,435, the entirety of which is incorporated hereinby reference), nitrilase, phytoene desaturase, aprotinin or a DNAsequence encoding Human Growth Hormone (U.S. Pat. No. 5,424,199, theentirety of which is incorporated herein by reference).

Plant cell plastids containing the constructs are also contemplated inthe invention, as are plants, plant seeds, plant cells or progenythereof containing plastids comprising the construct.

The present invention also includes methods for enhanced expression ofDNA sequences in plant plastids.

The invention also includes a method for the enhanced expression of anenzyme conferring herbicide tolerance in a plant cell, by expressing theAgrobacterium tumefaciens sp stain CP4 EPSPS in plastids of the plantcell.

In addition, the invention also includes a method for the enhancedexpression of an enzyme encoding hGH in plastids of the plant cell.

Thus, the present invention relates to a chimeric gene containing aherbicide tolerance coding sequence or the coding sequence of apharmaceutical protein, a plant plastid expression vector containing apromoter operably linked to a T7 Bacteriophage Polymerase gene 10ribosome binding site capable of enhanced expression in a plant plastidoperably linked to a herbicide tolerance or pharmaceutical coding gene,a plant transformation vector having inserted therein a herbicidetolerance or pharmaceutical coding gene expressed from a plastidpromoter linked to a T7 Bacteriophage Polymerase gene 10 ribosomebinding site, plant cells transformed using such vectors and plantsregenerated therefrom which exhibit a substantial degree of expressionof nucleic acid sequences and proteins and methods for producing suchplants and such plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence (SEQ ID NO:1&2) of the G10Lribosome binding site.

FIG. 2 provides an amino acid sequence (SEQ ID NO:4) encoding foraprotinin.

FIG. 3 provides the results of RP-HPLC analysis for characterization ofhGH protein expressed in the plastid. Peak I (tallest peak) indicatesthe expected retention time for properly folded, native 22 kDa GP2000.

FIG. 4 provides an electrospray ionization mass spectrometry (MS)analysis using a Micromass Q-T of electrospray time-of-flight massspectrometer. In particular, a series of ions corresponding to thespecie(s) present in the sample with varying numbers of protons attachedis provided. The axes of the spectrum are intensity versusmass-to-charge ratio of the specie(s) present.

FIG. 5 provides a graphic representation of the bioactivity of hGHexpressed from a plant plastid. The samples represented on the graph arebovine prolactin (bPL), hGH expressed from E. coli (Ala-hGH), and a nulltransgenic spiked with bovine prolactin (SPFF Null Spike) as positivecontrols, a null transgenic (SPFF Null) as a negative control, andtransgenic samples from a sepharose column (SPFF Sample, SPFF Sample)and a transgenic sample eluted from the sepharose column at pH3.5 (SPFFpH3.5 Eln).

FIG. 6 provides the nucleic acid sequence (SEQ ID NO:5) for thePrrn/G10L promoter/RBS hybrid. The Prrn promoter contains the consensusplastid −35 and −10 promoter elements (underlined) and the transcriptionstart sites (GC in bold) for the Plastid-Encoded RNA Polymerase (PEP).The gene 10 leader (G10L) contains a perfect plastid ribosome bindingsite (RBS, nucleotides in bold).

FIG. 7 provides the nucleic acid sequence (SEQ ID NOS:6) for thePrrn/NEP/G10L::14aaGFP fusion. The NEP promoter region is underlined (Ain bold is transcription start site). The NEP promoter region usedextends beyond the consensus sequence both upstream and downstream ofthe promoter. The initial ATG, the initiator methionine is not countedin the 14 amino acids (SEQ ID NO:7) of GFP.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, plastid expression constructsare provided which generally comprise a promoter functional in a plantplastid, a ribosome binding site derived from the T7 BacteriophagePolymerase gene 10 leader, a DNA sequence encoding for a gene ofinterest, and a transcription termination region capable of terminatingtranscription in a plant plastid. These elements are provided asoperably joined components in the 5′ to 3′ direction of transcription.

Furthermore, the constructs of the present invention may also include anucleic acid sequence encoding a peptide capable of targeting said DNAsequence encoding a protein to the thylakoid lumen within thechloroplast.

Of particular interest in the present invention is the use of theplastid expression constructs to direct the high level transcription andtranslation (expression) of nucleic acid sequences. Such plastidexpression constructs find use in directing the high level expression ofDNA sequences encoding for enzymes involved in herbicide tolerance orencoding for the production of pharmaceutical proteins.

Of more particular interest in the present invention is the use of theplastid expression constructs to direct the high level translation oftranscribed messenger RNA.

DNA sequence and biochemical data reveal a similarity of the plastidorganelle's transcriptional and translational machineries and initiationsignals to those found in prokaryotic systems. In fact, plastid derivedpromoter sequences have been reported to direct expression of reportergenes in prokaryotic cells. In addition, plastid genes are oftenorganized into polycistronic operons as they are in prokaryotes.

Despite the apparent similarities between plastids and prokaryotes,there exist fundamental differences in the methods used to control geneexpression in plastids and prokaryotes. As opposed to thetranscriptional control mechanisms typically observed in prokaryotes,plastid gene expression is controlled predominantly at the level oftranslation and mRNA stability by trans-acting nuclear encoded proteins.

Translation is a multi-stage process which first involves the binding ofmessenger RNA (mRNA) to ribosomes. Beginning at the translation startcodon, the mRNA codons are read sequentially as the ribosomes move alongthe mRNA molecule. The specified amino acids are then sequentially addedto the growing polypeptide chain to yield the protein or polypeptideencoded in the mRNA.

As mentioned, the first step in the translation process is the bindingof the mRNA molecule to the ribosome. The nature of this interaction(i.e. binding) has been only partially elucidated. Analysis ofRNase-resistant oligonucleotides isolated from bacterial translationinitiation complexes indicate that a RNA fragment approximately 30 to 40nucleotides in length comprises the initial ribosome binding site (RBS).Thus, a RBS is hereinafter understood to comprise a sequence of mRNAsurrounding the translation start codon which is responsible for thebinding of the ribosome and for initiation of translation.

Recently, ribosome binding sites have been identified capable ofdirecting translation in a prokaryotes. For example, a ribosome bindingsite derived from the T7 bacteriophage gene 10 leader, G10L (U.S. Pat.No. 5,232,840, the entirety of which is incorporated herein byreference), has been identified which enhances expression of nucleicacid sequences in prokaryotes.

Herbicides such as N-phosphonomethylglycine, halogenatedhydroxybenzonitriles, and norflurazon have been the subject of a largeamount of investigation.

N-phosphonomethylglycine, commonly referred to as glyphosate, inhibitsthe shikimic acid pathway which leads to the biosynthesis of aromaticcompounds including amino acids, plant hormones and vitamins.Specifically, glyphosate curbs the conversion of phosphoenolpyruvic acid(PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acidby inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(hereinafter referred to as EPSP synthase or EPSPS).

Glyphosate tolerant plants have been produced by transformation ofvarious EPSP synthase genes into the nuclear genome of a plant. A genefor EPSP synthase has been cloned from Agrobacterium tumefaciens spstrain CP4 (U.S. Pat. No. 5,633,435) and confers a high level ofglyphosate tolerance in plants. Furthermore, high levels of glyphosatetolerance has been achieved in a number of crop plants by fusing EPSPSto a chloroplast transit peptide (CTP) for targeted expression inplastids. In addition, variants of the wild-type EPSPS enzyme have beenisolated which are glyphosate tolerant as a result of alterations in theEPSPS amino acid coding sequence (Kishore and Shah, Ann. Rev. Biochem.(1988) 57:627-663; Shulze et al., Arch. Microbiol. (1984) 137:121-123;Kishore et al., Fed. Proc. (1986) 45:1506). These variants typicallyhave a higher K_(i) for glyphosate than the wild-type EPSPS enzyme whichconfers the glyphosate tolerant phenotype, but these variants are alsocharacterized by a high K_(m) for PEP which makes the enzyme kineticallyless efficient (Kishore and Shah, Ann. Rev. Biochem. (1988) 57:627-663;Sost et al., FEBS Lett. (1984) 173:238-241; Shulze et al., Arch.Microbiol. (1984) 137:121-123; Kishore et al., Fed. Proc. (1986)45:1506; Sost and Amrhein, Arch. Biochem. Biophys. (1990) 282:433-436).

In addition to engineering plants for glyphosate tolerance, plants havealso been engineered to tolerate other classes of herbicides such ashalogenated hydroxybenzonitriles, and norflurazon using nucleic acidsequences expressed in the nucleus.

Halogenated hydroxybenzonitriles, such as Bromoxynil, are suggested toact herbicidally by inhibiting the quinone-binding protein complex ofphotosystem II, inhibiting electron transfer (Van Rensen (1982) Physiol.Plant 54:515-520,and Sanders and Pallett (1986) Pestic. Biochem.Physiol. 26:116-122). Herbicides such as norflurazon inhibit theproduction of carotenoids.

Plants which are resistant to Bromoxynil have been produced byexpressing DNA sequences encoding for enzymes capable of detoxifyingBromoxynil (nitrilases) in the plant cell nucleus. DNA sequencesencoding for such nitrilases have been cloned from bacteria such asKlebsiella pneumoniae and used to construct vectors to direct theexpression of the DNA sequence in plant cell nucleus (U.S. Pat. No.4,810,648, the entirety of which is incorporated herein by reference).

Plants which are resistant to Norflurazon have been engineered byexpressing nucleic acid sequences which encode for enzymes in thecarotenoid biosynthetic pathway in plant cell nuclei. For example, byexpressing a phytoene desaturase from Erwinia uredovora providestolerance to norflurazon.

While plants transformed to express nucleic acid sequences encoding forsuch enzymes from the nuclear genome have found utility in engineeringherbicide tolerant plants, it would be increasingly beneficial to obtainherbicide tolerant plants via plastidial expression.

In the examples provided herein, DNA sequences encoding for enzymesinvolved in herbicide tolerance are used in constructs to direct theexpression of the sequences from the plant plastid. DNA sequencesencoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS),bromoxynil nitrilase (Bxn), phytoene desaturase (crtI (Misawa et al,(1993) Plant Journal 4:833-840, and (1994) Plant Jour 6:481-489), andacetohydroxyacid synthase (AHAS (Sathasiivan et al. (1990) Nucl. AcidsRes. 18:2188-2193)) are used in the expression constructs of the presentinvention to direct the expression of said herbicide tolerancenucleotide sequences from the plant plastid.

Transplastomic tobacco plants are identified which are homoplasmic forthe DNA sequences of interest encoding said herbicide tolerance genes.Homoplasmic plants demonstrate a high level of protein expression fromthe plastid. Furthermore, homoplasmic plants demonstrate a high level oftolerance for the respective herbicide. For example, as described inmore detail in the example below, plants transformed to express EPSPSfrom the plastid demonstrate a high level of tolerance for the herbicideglyphosate. In addition, homoplasmic tobacco lines expressing nitrilaseor phytoene desaturase demonstrate high levels of tolerance for theherbicides bromoxynil and norflurazon, respectively.

An artisan skilled in the art to which the present invention pertainswill recognize that additional sequences may be employed to in theplastid expression constructs of the instant invention to produceherbicide tolerant plants. Other nucleic acid sequence which may finduse in the plastid expression constructs herbicide tolerant plantsinclude the bar gene for tolerance to glufosinate (DeBlock, et al.(1987) EMBO J. 6:2513-2519).

Furthermore, additional glyphosate tolerance genes may be employed inthe constructs of the present invention. Additional glyphosate tolerantEPSPS genes are described in U.S. Pat. No. 5,627,061, Padgette et al.(1996) Herbicide Resistant Crops, Lewis Publishers, 53-85, and inPenaloza-Vazquez, et al. (1995) Plant Cell Reports 14:482-487, theentireties of which are incorporated herein by reference.

It should be noted that the herbicide tolerance constructs of thepresent invention may also include sequences encoding genes involved inother stress tolerance genes, for example insect or diseaseresistance/tolerance genes. As described in more detail in the examplesthat follow, plastid expression constructs are used to regenerate plantswhich are resistant to the herbicide Buctril, which also expresses theBacillus thuringensis cry1Ac protein.

In addition, the plastid expression constructs also find use indirecting the production of human biological proteins (pharmaceuticalproteins) from the plant plastid. Nucleic acid sequences encoding forthe Human Growth Hormone (hGH) are employed in the plastid expressionconstructs of the present invention. Furthermore, transplastomic tobaccoplants containing such constructs demonstrate a high level of expressionof hGH. In addition, the hGH protein expressed from the plant plastidexhibits characteristics of proper processing as well as proper proteinfolding.

Traditional methods of pharmaceutical protein production generallyemploy prokaryotic or single cell eukaryotic organisms for expressionand large scale production systems. For example, production of the humanbiologic, Human Growth Hormone (U.S. Pat. No. 5,424,199), has beenachieved in Bacillus and E. coli cells.

Another example is the production of aprotinin. Traditional methods forthe production of aprotinin have employed the expression of aprotinin inbacteria, or more usually, the extraction of the protein from bovineorgans or tissues. Thus, there is a need in the art for an alternativeapproach for the large scale production of such human biologics.

Human Growth Hormone (hGH) participates in much of the regulation ofnormal human growth and development. This 22,000 dalton pituitaryhormone exhibits a multitude of biological effects including lineargrowth (somatogenesis), lactation, activation of macrophages,insulin-like and diabetogenic effects among others (Chawla, Ann. Rev.Med. (1983) 34:519; Edwards, et al., Science (1988) 239:769; Thorner etal., J. Clin. Invest. (1988) 81:745). Growth deficiency in childrenleads to dwarfism, which has been successfully treated for more than adecade by exogenous administration of hGH. hGH is a member of a familyof homologous hormones that include placental lactogens, prolactins, andother genetic and species variants or growth hormone (Nicoll, et al.,Endocrine Reviews (1986) 7:169). hGH is unusual among these in that itexhibits broad species specificity and binds to either the clonedsomatogenic (Leung, et al., Nature (1987) 33:537) or prolactin receptor(Boutin, et al., Cell (1988) 53:69).

Aprotinin (also known as bovine pancreatic trypsin inhibitor, BPTI) is abasic protein present in several bovine organs and tissues, such as thelymph nodes, pancreas, lungs, parotid gland, spleen and liver.

Aprotinin is known to inhibit various serine proteases, includingtrypsin, chymotrypsin, plasmin and kallikrein, and is usedtherapeutically in the treatment of acute pancreatitis, various stagesof shock syndrome, hyperfibrinolytic hemorrhage and myocardialinfarction. In addition, administration of aprotinin in high dosessignificantly reduces blood loss in connection with cardiac surgery,including cardiopulmonary bypass (Bidstrup, et al. (1989) CardiovascSurg. 44:640-645).

As demonstrated in more detail in the examples that follow, plastidexpression constructs are employed to direct the expression of aprotininand a human growth hormone from the plant plastid.

Other sequences which may find use in the production of human biologicsinclude sequences encoding for insulin or insulin precursors may finduse in the expression constructs of the present invention. The skilledartisan will recognize that many nucleotide sequences encoding for humanbiologics may be employed in the constructs of the present invention todirect their expression from a plant plastid such as those described inGoodman and Gelman (1990) Pharmacological Basis of Therapeutics,Pergaman Press, 8^(th) Edition, Sections 14 and 15.

In developing the constructs the various fragments comprising theregulatory regions and open reading frame may be subjected to differentprocessing conditions, such as ligation, restriction enzyme digestion,PCR, in vitro mutagenesis, linkers and adapters addition, and the like.Thus, nucleotide transitions, transversions, insertions, deletions, orthe like, may be performed on the DNA which is employed in theregulatory regions or the DNA sequences of interest for expression inthe plastids. Methods for restriction digests, Klenow blunt endtreatments, ligations, and the like are well known to those in the artand are described, for example, by Maniatis et al. (in Molecularcloning: a laboratory manual (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

During the preparation of the constructs, the various fragments of DNAwill often be cloned in an appropriate cloning vector, which allows foramplification of the DNA, modification of the DNA or manipulation of theDNA by joining or removing sequences, linkers, or the like. Preferably,the vectors will be capable of replication to at least a relatively highcopy number in E. coli. A number of vectors are readily available forcloning, including such vectors as pBR322, vectors of the pUC series,the M13 series vectors, and pBluescript vectors (Stratagene; La Jolla,Calif.).

In order to provide a means of selecting the desired plant cells,vectors for plastid transformation typically contain a construct whichprovides for expression of a selectable marker gene. Marker genes areplant-expressible DNA sequences which express a polypeptide whichresists a natural inhibition by, attenuates, or inactivates a selectivesubstance, i.e., antibiotic, herbicide etc.

Alternatively, a marker gene may provide some other visibly reactiveresponse, i.e., may cause a distinctive appearance or growth patternrelative to plants or plant cells not expressing the selectable markergene in the presence of some substance, either as applied directly tothe plant or plant cells or as present in the plant or plant cell growthmedia.

In either case, the plants or plant cells containing such selectablemarker genes will have a distinctive phenotype for purposes ofidentification, i.e., they will be distinguishable from non-transformedcells. The characteristic phenotype allows the identification of cells,cell groups, tissues, organs, plant parts or whole plants containing theconstruct.

Detection of the marker phenotype makes possible the selection of cellshaving a second gene to which the marker gene has been linked. Thissecond gene typically comprises a desirable phenotype which is notreadily identifiable in transformed cells, but which is present when theplant cell or derivative thereof is grown to maturity, even underconditions wherein the selectable marker phenotype itself is notapparent.

The use of such a marker for identification of plant cells containing aplastid construct has been described by Svab et al. (1993, supra). Inthe examples provided below, a bacterial aadA gene is expressed as themarker under the regulatory control of chloroplast 5′ promoter and 3′transcription termination regions, specifically the regulatory regionsof thepsbA gene (described in Staub et al., EMBO J. (1993)12(2):601-606). Numerous additional promoter regions may also be used todrive expression of the selectable marker gene, including variousplastid promoters and bacterial promoters which have been shown tofunction in plant plastids.

Expression of the aadA gene confers resistance to spectinomycin andstreptomycin, and thus allows for the identification of plant cellsexpressing this marker. The aadA gene product allows for continuedgrowth and greening of cells whose chloroplasts comprise the selectablemarker gene product. Cells which do not contain the selectable markergene product are bleached. Selection for the aadA gene marker is thusbased on identification of plant cells which are not bleached by thepresence of streptomycin, or more preferably spectinomycin, in the plantgrowth medium.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes which encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, geneswhich provide resistance to plant herbicides such as glyphosate,bromoxynil or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al., J. Biol. Chem. (1985) 260:4724-4728(glyphosate resistant EPSP); Stalker et al., J. Biol. Chem. (1985)263:6310-6314 (bromoxynil resistant nitrilase gene); and Sathasivan etal., Nucl. Acids Res. (1990) 18:2188 (AHAS imidazolinone resistancegene)).

Stable transformation of tobacco plastid genomes by particle bombardmentis reported (Svab et al. (1990), supra) and Svab et al. (1993), supra).The methods described therein may be employed to obtain plantshomoplasmic for plastid expression constructs.

Generally, bombarded tissue is cultured for approximately 2 days on acell division-promoting media, after which the plant tissue istransferred to a selective media containing an inhibitory amount of theparticular selective agent, as well as the particular hormones and othersubstances necessary to obtain regeneration for that particular plantspecies. Shoots are then subcultured on the same selective media toensure production and selection of homoplasmic shoots.

Transplastomic tobacco plants are analyzed for a pure population oftransformed plastid genomes (homoplasmic lines). Homoplasmy is verifiedusing Southern analysis employing nucleic acid probes spanning a regionof the transgene and chloroplast genome (i.e. the insertion region).Transplastoimc plants which are heteroplasmic (i.e. contain a mixture ofplastid genomes containing and lacking the transgene) are characterizedby a hybridization pattern of wild type and transgenic bands.Homoplasmic plants show a hybridization pattern lacking the wild typeband.

Alternatively, homoplasmy may be verified using the polymerase chainreaction (PCR). PCR primers are utilized which are targeted to amplifyfrom sequences from the insertion region. For example, a pair of primersmay be utilized in a PCR reaction. One primer amplifies from a region inthe transgene, while the second primer amplifies from a region proximalto the insertion region towards the insertion region. A second PCRreaction is performed using primers designed to amplify the region ofinsertion. Transplastomic lines identified as homoplasmic produce theexpected size fragment in the first reaction, while they do not producethe predicted size fragment in the second reaction.

Where transformation and regeneration methods have been adapted for agiven plant species, either by Agrobacterium-mediated transformation,bombardment or some other method, the established techniques may bemodified for use in selection and regeneration methods to produceplastid-transformed plants. For example, the methods described hereinfor tobacco are readily adaptable to other solanaceous species, such astomato, petunia and potato.

For transformation of soybean, particle bombardment as well asAgrobacterium-mediated nuclear transformation and regeneration protocolshave been described (Hinchee et al. U.S. Pat. No. 5,416,011, andChristou et al. U.S. Pat. No. 5,015,580). The skilled artisan willrecognize that protocols described for soybean transformation may beused

In Brassica, Agrobacterium-mediated transformation and regenerationprotocols generally involve the use of hypocotyl tissue, a non-greentissue which might contain a low plastid content. Thus, for Brassica,preferred target tissues would include microspore-derived hypocotyl orcotyledonary tissues (which are green and thus contain numerousplastids) or leaf tissue explants. While the regeneration rates fromsuch tissues may be low, positional effects, such as seen withAgrobacterium-mediated transformation, are not expected, thus it wouldnot be necessary to screen numerous successfully transformed plants inorder to obtain a desired phenotype.

For cotton, transformation of Gossypium hirsutum L. cotyledons byco-cultivation with Agrobacterium tumefaciens has been described byFiroozabady et al., Plant Mol. Bio. (1987) 10:105-116 and Umbeck et al.,Bio/Technology (1987) 5:263-266. Again, as for Brassica, this tissue maycontain insufficient plastid content for chloroplast transformation.Thus, as for Brassica, an alternative method for transformation andregeneration of alternative target tissue containing chloroplasts may bedesirable, for instance targeting green embryogenic tissue.

Other plant species may be similarly transformed using relatedtechniques. Alternatively, microprojectile bombardment methods, such asdescribed by Klein et al. (Bio/Technology 10:286-291) may also be usedto obtain nuclear transformed plants comprising the viral single subunitRNA polymerase expression constructs described herein. Cottontransformation by particle bombardment is reported in WO 92/15675,published Sep. 17, 1992. Suitable plants for the practice of the presentinvention include, but are not limited to, soybean, cotton, alfalfa, oilseed rape, flax, tomato, sugar beet, sunflower, potato, tobacco, maize,wheat, rice and lettuce.

The vectors for use in plastid transformation preferably include meansfor providing a stable transfer of the plastid expression construct andselectable marker construct into the plastid genome. This is mostconveniently provided by regions of homology to the target plastidgenome. The regions of homology flank the construct to be transferredand provide for transfer to the plastid genome by homologousrecombination, via a double crossover into the genome. The complete DNAsequence of the plastid genome of tobacco has been reported (Shinozakiet al., EMBO J. (1986) 5:2043-2049). Complete DNA sequences of theplastid genomes from liverwort (Ohyama et al., Nature (1986)322:572-574) and rice (Hiratsuka et al., Mol. Gen. Genet. (1989)217:185-194), have also been reported.

Where the regions of homology are present in the inverted repeat regionsof the plastid genome (known as IRA and IRB), two copies of thetransgene are expected per transformed plastid. Where the regions ofhomology are present outside the inverted repeat regions of the plastidgenome, one copy of the transgene is expected per transformed plastid.The regions of homology within the plastid genome are approximately 1 kbin size. Smaller regions of homology may also be used, and as little as100 bp can provide for homologous recombination into the plastid genome.However, the frequency of recombination and thus the frequency ofobtaining plants having transformed plastids decreases with decreasingsize of the homology regions.

Examples of constructs having regions of homology the plastid genome aredescribed in Svab et. al. (1990 supra), Svab et al. (1993 supra) andZoubenko et al. (Nuc Acid Res (1994) 22(19):3819-3824).

As described in more detail in the examples below, constructs aredescribed which provide for enhanced expression of DNA sequences inplant plastids. Various promoter/ribosome binding site sequences areemployed to direct expression in plant plastids. Promoter sequences ofthe 16S ribosomal RNA operon (Prrn) are linked to a ribosome bindingsite (RBS) derived from the T7 bacteriophage gene 10 leader sequence(G10L). DNA sequences expressed under the regulatory control of thePrrn/G10L sequence show a significantly higher level of proteinexpression than those levels obtained under the control of otherpromoter/RBS combinations, while expression of mRNA may or may not behigher in these plants.

In the examples below, nucleic acid sequences encoding CP4 EPSP synthase(U.S. Pat. No. 5,633,435) are placed into expression constructs forexpression of EPSP synthase enzyme from the plant plastid. Furthermore,a DNA sequence encoding for hGH (U.S. Pat. No. 5,424,199) is also placedinto expression construct for the expression of human growth hormonefrom the plant plastid. The constructs prepared utilize a ribosomebinding site designed after the T7 bacteriophage gene 10 leader (G10L)to increase the expression of the nucleic acid sequences in the plantplastid.

Plastid expression constructs encoding for the expression of EPSPS andhGH are introduced via a chloroplast transformation vector.

Tobacco lines containing the native encoding sequence to the EPSPSenzyme expressed in plastids under the control of the Prrn/G10Lpromoter/ribosome binding site sequence demonstrate a significantlyhigher level of protein expression than those levels obtained from EPSPSexpressed under the control of the Prrn/rbcL RBS sequence. However,EPSPS mRNA is expressed at a higher level in plants expressing CP4 EPSPSfrom the plastid under the control of the Prrn/rbcL(RBS). These resultsindicate that translation from transcripts containing the T7bacteriophage gene 10 ribosome binding site is more efficient. Inaddition, protein expression levels of EPSPS obtained fromtransplastomic tobacco lines expressing EPSPS under the control of thePrrn/G10L RBS provide for a high level of glyphosate tolerance.

Furthermore, transplastomic tobacco lines transformed to express hGHunder the control of the Prrn/G10L promoter/ribosome binding sitesequence demonstrate a significantly higher level of protein expressionthan those levels obtained from hGH expressed under the control of thePpsbA promoter/RBS sequence.

Increases in protein expression levels of at least approximately 200fold may be obtained from constructs utilizing Prrn/G10L ribosomebinding site for expression of EPSPS and hGH over the expression levelsobtained from other promoter/RBS combinations for plastid expression. Inaddition, protein levels obtained from plastid expression constructsutilizing the Prrn/G10L promoter/RBS sequence may accumulate 50 to 3500fold higher levels than from nuclear expression constructs. Thus,inclusion of the G10L ribosome binding site in plastid expressionconstructs may find use for increasing the levels of protein expressionfrom plant plastids.

Furthermore, the constructs of the present invention may also includesequences to target the expressed protein to a particular suborganellarregion, for example, the thylakoid lumen of the chloroplast. Forexample, as described in the examples below, a nucleotide sequenceencoding a peptide from the plastid genome cytochrome f targets theexpressed aprotinin protein to the thylakoid membrane. Such targeting ofexpressed proteins may provide for a compartmentalization of the proteinallowing for increased oxidative stability and proper protein folding.

Thus, the constructs and methods of the present invention provide ameans for obtaining transplastomic plants with high level tolerance ofherbicides. High levels of tolerance include tolerance of vegetativetissue when amounts of greater than about 16 oz/acre glyphosate areapplied, preferably greater than about 32 oz/acre, more preferablygreater than about 64 oz/acre, most preferably greater than about 128oz/acre. Furthermore, high levels of tolerance can also includetolerance of reproductive tissues when amounts of greater than about 16oz/acre glyphosate are applied, preferably greater than about 32oz/acre, most preferably greater than about 64 oz/acre.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES Example 1 Expression Constructs

Constructs and methods for use in transforming the plastids of higherplants are described in Zoubenko et al. (Nuc Acid Res (1994)22(19):3819-3824), Svab et al. (Proc. Natl. Acad. Sci. (1990)87:8526-8530 and Proc. Natl. Acad. Sci. (1993) 90:913-917) and Staub etal. (EMBO J. (1993) 12:601-606). Constructs and methods for use intransforming plastids of higher plants to express DNA sequences underthe control of a nuclearly encoded, plastid targeted T7 polymerase aredescribed in U.S. Pat. No. 5,576,198. The complete DNA sequences of theplastid genome of tobacco are reported by Shinozaki et al. (EMBO J.(1986) 5:2043-2049). All plastid DNA references in the followingdescription are to the nucleotide number from tobacco.

The complete nucleotide sequence encoding the tobacco cytochrome f(petA)is described in Bassham et al, (1991) J Biol Chem 266:23606-23610and Konishi et al. (1993) Plant Cell Physiol 34:1081-1087.

1A. Promoter/Ribosome Binding Site Sequences

The promoter region of the plastid 16S ribosomal RNA operon (Prrn) islinked to a synthetic ribosome binding site (RBS) patterned on theplastid rbcL gene leader to create the Prrn/rbcLRBS fragment. ThePrrn/rbcLRBS sequence is as described in Svab et al. (1993, supra) forthe Prrn/rbcL(S) fragment.

The promoter region of the plastid psbA promoter (PpsbA) and terminatorsequences (TpsbA) are described in Staub et al. (1993, EMBO J., 12,601-606).

The Prrn/G10L sequence was constructed by annealing two oligonucleotidesequences, T71ead1 and T71ead2 (Table 1), to create the G10L plastidribosome binding site (FIG. 1). The G10L sequence was ligated to the 3′terminus of the Prrn promoter sequence as an EcoRI/NcoI fragment tocreate the Prrn/G10L sequence.

TABLE 1 T7lead1 5′-AAT TGT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GATATA CC-3′ (SEQ ID NO:1) T7lead2 5′-CAT GGG TAT ATC TCC TTC TTA AAG TTAAAC AAA ATT ATT TCT AC-3′ (SEQ ID NO:3)

Chimeric genes are preferably inserted into the expression vector todirect their transcription from the Prrn promoter. Thus, in the plastidgenome, chimeric genes are transcribed from the Prrn/RBS promoter, orthe Prrn/G10L promoter in the plant plastid. The nucleic acid sequenceof the Prrn/G10L fusion is provided in FIG. 6.

1B. CP4 EPSPS Plastid Expression Constructs

A plastid expression vector pMON30117 is constructed from a precursorvector pPRV111B (Zoubenko, et al. 1994, supra, GenBank accessionU12813). The vector pMON30117 carries a multiple cloning site forinsertion of a passenger gene in a Prrn/rbcLRBS/Trps16 expressioncassette. The Prrn/rbcLRBS sequence is cloned into pPRV111B vector as anEcoRI/NcoI fragment, and the terminator region from the plastid rps16gene(Trps16) is cloned 3′ of the Prrn promoter as a HindIII/NcoIfragment. The Trps16 fragment comprises the rps16 gene 3′-regulatoryregion from nucleotides 5,087 to 4,939 in the tobacco plasmid DNA.

The pPRV111B backbone of the vector pMON30117 contains a marker gene,aadA, for selection on spectinomycin and streptomycin, and rps 7/12 forthe integration, by homologous recombination, of the passenger DNA intotrnV-rps7/12 intergenic region.

A nuclear expression construct, pMON10154, was prepared as a control forintegration into plants by Agrobacterium-mediated transformation. Inthis construct, the CP4 native gene is expressed from the constitutiveFigwort Mosaic Virus promoter and the Petunia HSP70 leader, and has theE9 terminator. Targeting to plastids is by the chloroplast transitpeptide of the Petunia EPSPS translationally fused to the N-terminus ofthe CP4 gene.

The plastid expression construct pMON30118 was prepared by cloning thenative CP4 EPSPS gene fused with the N-terminal five (5) amino acidsfrom the plastid rbcL (described in Svab et al., 1993 supra) gene as anNcoI/SmaI fragment into the multiple cloning site of the vectorpMON30117.

The plastid expression construct pMON30123 is essentially the same aspMON30118 with the exception of the deletion of the N-terminal five (5)amino acids from the plastid rbcL.

The plastid expression construct pMON30130 was created by replacing thenative CP4 EPSPS of pMON30123, with a synthetic CP4 gene. This constructalso lacks the N-terminal 5 amino acid fusion from the plastid rbcLgene.

The plastid expression construct pMON38773 was constructed by replacingthe Prrn/RBS sequence of pMON30123 with the Prrn/G10L promoter sequencedescribed above. The EPSPS DNA sequence of pMON38773 also lacks theN-terminal 5 amino acid fusion from the plastid rbcL gene.

A plastid expression construct, pMON38766 was constructed using thepromoter from T7 phage gene 10 (P-T7), including G10L, CP4 (native) genecoding region, and the terminator sequence from plastid rps16 gene(Trps16).

A plastid expression construct, pMON38797 was constructed using thepromoter from T7 phage gene 10 (P-T7), including G10L, CP4 (synthetic)gene coding region, terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON38798 was constructed using thepromoter of the 16SrDNA operon (Prrn), G10L, CP4 (synthetic) gene codingregion, terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON38793 was constructed using thepromoter of the 16SrDNA operon (Prrn), a synthetic ribosome binding site(RBS) patterned from the plastid rbcL gene, the glyphosate tolerantPetunia EPSP synthase gene (P-EPSPS, Padgette, et al. (1987) Arch.Biochem. Biophys. 258:564-573) carrying the mutation Glycine to Alanineat amino acid position 101, terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON38796 was constructed using thepromoter of the 16SrDNA operon (Prrn), synthetic ribosome binding site(RBS) patterned from the plastid rbcL gene, the glyphosate tolerantAchromobacter (strain LBAA) EPSP synthase gene (U.S. Pat. No. 5,627,061,the entirety of which is incorporated herein by reference)carrying themutation Glycine to Alanine at amino acid position 100 (G100A),terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON45204, was constructed using thepromoter of the 16SrDNA operon (Prrn) with the G10L, the glyphosatetolerant Pseudomonas (strain LBAA) EPSP synthase gene carrying themutation Glycine to Alanine at amino acid position 100 (G100A),terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON45201, was constructed using thepromoter of the 16SrDNA operon (Prrn), synthetic ribosome binding site(RBS) patterned from the plastid rbcL gene, wild-type glyphosatetolerant Bacillus subtilis aroE (EPSPS)(U.S. Pat. No. 5,627,061) gene,terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON45259, was constructed using thepromoter of the 16SrDNA operon (Prrn) with the G10L sequencefunctionally associated with the nucleic acid sequence encoding thesynthetic CP4 protein having an additional sequence at the N-terminusencoding the first 14 amino acids of the green fluorescent protein (GFP)(GKGEELFTGVVPSM). The sequence encoding the 14 amino acid GFP fusionbegins at the glycine in the second position of the protein. Theconstruct also contains the rps16 terminator.

Another plastid expression construct, pMON49218, was constructed toexpress the synthetic CP4 sequence with the 14 amino acid GFP fusionfrom the promoter region of the 16SrDNA operon having thenuclear-encoded RNA polymerase region (PrrnPEP+NEP), and the terminatorregion from the plastid rps16 gene. The DNA sequence of thePrrn/NEP/G10L::14aaGFP fusion is provided in FIG. 7.

1C. Bucril (bxn) Plastid Expression Constructs

The bxn herbicide resistance gene (U.S. Pat. No. 4,810,648, the entiretyof which is incorporated herein by reference) was removed from theplasmid pBrx47 as an Nco I to Asp718 restriction fragment and clonedinto Nco/Asp718 cut pUC120 resulting in plasmid pBrx87. Plasmid pBrx87was then digested with Nco/Xba and cloned into the Nco/Xba sites of theplasmid pLAA21 which contains the Prrn plastid promoter and the rpsL 3′region for plastid expression. The resulting plasmid was designatedpBrx89. Plasmid pBrx89 was digested with Sac I and Hind III and the 1.5kb chimeric bxn gene with plastid expression signals was inserted intothe Sac/Hind III sites of the tobacco plastid homology vector pOVZ44B(Zoubenko et al, Nuc Acids Res 22: 3819-3824 (1994)) to create plasmidpCGN5175.

To construct plasmid pCGN6114, plasmid pBrx90 (a Bluescript plasmidcontaining the bxn gene encoding the bromoxynil specific nitrilase) wasdigested with Nco I/Asc I and the bxn structural gene was substitutedfor the GUS gene in the Nco/Asc digested plasmid pCGN5063 resulting inplasmid pCGN6107. This plasmid contains the the bxn gene under thecontrol of the T7 promoter/gene10 leader at the 5′ end and the psbA/T7hybrid transcriptional terminator at the 3′ end of the chimeric gene.This T7 promoter/bxn chimeric gene was excised from pCGN6107 as a HindIII/Not I DNA segment and moved into the choromphenical plasmidBCSK+(Stratagene) at the Hind III/Not sites to create plasmid pCGN6109.The chimeric gene was them moved as a Hind III/Not fragment frompCGN6109 into the chloroplast homology vector pOVZ44B described above tocreate plasmid pCGN6114. Tobacco plants transformed with pCGN6114require the T7 RNA polymerase be provided in the plant plastidbackground to activate transcription of the chimeric bxn gene via the T7promoter. This system has previously been detailed in McBride et al PNAS91:7301-7305 (1994) and McBride et al U.S. Pat. No. 5,576,198.

1D. BXN/AHAS Plastid Expression Constructs

A plastid expression construct, pCGN5026, is prepared to direct theexpression of BXN and AHAS from the plant plastid. The AHAS nucleotidesequence (described in EP Publication Number 0 525 384 A2, the entiretyof which is incorporated herein by reference) is translationally linkedto the BXN nucleotide sequence (U.S. Pat. No. 4,810,648, the entirety ofwhich is incorporated herein by reference). The AHAS structural geneencoding acetohydroxyacid synthase was cloned from the plasmid pCGN4277as an Nco I to Age DNA fragment into the Nco/Xma sites of plasmid pUC120to create plasmid pCGN5022. This plasmid was then digested with theenzymes BamH I and Pst and a 1.3 kb Bam/Pst DNA segment containing thebxn gene encoding the bromoxynil-specific nitrilase was excised from theplasmid pBrx26 and cloned into the Bam/Pst sites of pCGN5022 to createplasmid pCGN5023. Plasmid pCGN5023 contained a 3.3 kb DNA segmentcontaining the AHAS/bxn operon segment and this fragment. This plasmidwas cut at the unique Pst site and this Pst site was removed andreplaced with a synthetic linker containing a unique Xba I restrictionsite generating plasmid pCGN5024. Plasmid pCGN5024 was digested withNco/Xba and the 3.3 kb Nco/Xba DNA fragment was cloned into the plastidpromoter cassette vector pLAA21 (Pst) that had been digested with Ncoand Xba to remove the GUS gene. The plasmid resulting from this cloningwas designated plasmid pCGN5025 and contained the herbicide operon underthe control of the plastid promoter Prrn and the rpsL 3′ DNA segment.The entire chimeric herbicide operon under the control of the plastidexpression elements was excised from pCGN5025 as a Sac I/Pst DNAfragment and cloned into the Sac/Pst sites of the plastid homologycassette vector pOVZ44B (Zoubenko et al, Nuc Acids Res 22:3819-3824(1994)) to facilitate transfer into the tobacco chloroplast genome.

1E. Bt cry1Ac and bxn Plastid Expression Construct

Plasmid pBrx9 (Stalker and McBride, (1987) J Bacteriol 169:955-960), anoriginal clone from Klebsiella containing a bxn gene DNA segment, wasused as a template to generate an ˜450 bp BamH I/Cla I PCR DNA fragmentthat encompasses the N-terminal end of the bxn gene and includes 44 bpof the 5′ untranslated portion of the native gene. This fragment wasexchanged with the ˜400 bp Bam/Cla fragment in the plasmid pBrx90resulting in plasmid pBrx90.1. This plasmid contains the entire bxn geneand the 44 bp untranslated 5′ DNA segment. The bxn gene was excised fromplasmid pBrx90.1 as a Bam/Asc I DNA segment and inserted into plasmidpCGN5146 at the Bgl II/Asc I sites to generate plasmid pCGN5191. PlasmidpCGN5146 is a pKK233-2 (Pharmacia) derivative containing the full-lengthcry1Ac gene encoding the HD-73 Bt protoxin. Plasmid pCGN5191 thencontains the cry1Ac and bxn genes in an operon configuration with thebxn gene being the distal gene in the operon. Both genes are under thecontrol of the Ptac promoter for E coli expression in 5191. PlasmidpCGN5191 was digested with Nco/Asc and the Nco/Asc DNA fragmentcontaining the Bt/bxn operon was cloned into the Nco/Asc sites of thechloroplast homology vector pCGN5155, a derivative of pOVZ44B. Theresulting plasmid, pCGN5197 contains the Bt/bxn operon under the controlof the Prrn plastid promoter and rpsL transcription terminator regions.This plasmid facilitated transfer of the Bt/bxn chimeric operon into thetobacco plastid genome.

1F. Phytoene Desaturase Plastid Expression Constructs

The crtI gene was obtained as a Hind III/Sal I PCR fragment from theoriginal plasmid containing the Erwinia carotova crt operon (Misawa etal, (1994) Plant Jour 6:481-489)) and cloned as a Hind III/Sal DNAsegment into BCSK+(Stratagene) at the Hind III/Sal sites to generateplasmid pCGN5172. The crtI fragment was cloned from pCGN5172 as an NcoI/Sal I fragment into pCGN5038 (a derivative of pOVZ44B) to create theplastid expression construct pCGN5177. This construct directs theexpression of the crtI sequence from the Prrn promoter and the rps16terminator sequence. This plasmid facilitated the transfer of thechimeric crtI gene into the tobacco plastid genome.

1G. hGH Expression Constructs for Plant Transformation

Nuclear Expression Constructs

The construct pWRG4747 was constructed to direct the expression of hGHin the plant nuclear genome. This vector contains the hGH operablylinked to the Figwort Mosaic Virus promoter (U.S. Pat. No. 5,378,619,the entirety is incorporated herein by reference) and the CTP2 leaderfor directing the hGH protein into the plastid. The FMV/CTP2L::hGH::NpAfragment is cloned along with the DNA sequence conferring resistance toKanamycin between the right and left borders (RB and LB) of the transferDNA (tDNA) of Agrobacterium tumefaciens to direct the integration intothe nuclear genome.

The nuclear transformation vector pWRG4744 contains essentially the sameelements as pWRG4747 except the construct lacks the CTP2 leader and thehGH protein is directed to the plant cell cytoplasm.

Plastid Expression Constructs

The plastid expression vector pWRG4838 was constructed using the fulllength hGH gene expressed from the promoter region from the psbA geneand the psbA gene terminator, PpsbA and TspbA respectively (described inStaub et al. (1993), supra). This chimeric promoter-gene-terminatorfusion (PpsbA::hGH::TpsbA) is cloned adjacent to the selectable markergene aadA also driven by the plastid expression elements of the psbAgene. The two chimeric gene sequences are cloned into a vector betweentwo sequences which direct the integration of the chimeric genesequences into the tobacco plastid genome upstream of the plastid16SrDNA. This is joined to a 1 kb Ampicillin resistance gene whichprovides for selection of E. coli containing the construct and the pUCorigin of replication for plasmid maintenance in E. coli.

The plastid expression construct pMON38755 was prepared using the hGHDNA sequence translationally fused at the N-terminus with the yeastubiquitin gene, creating the Ubi-hGH fusion gene. The Ubi-hGH fusiongene is cloned next to the aadA gene for selection of transplastomictobacco on media containing spectinomycin or streptomycin (from pPRV112Bdescribed in Zoubenko et al. (1994) supra). Sequences are included forthe homologous recombination of sequences encoding for hGH and aadAexpression. These sequences are obtained from the vector pPRV112Bdescribed in Zoubenko et al. (1994, supra). This is joined to a 1 kbAmpicillin resistance gene which provides for selection of E. colicontaining the construct and the pUC origin of replication for plasmidmaintenance in E. coli.

The plastid expression construct pMON38794 contains essentially the sameelements as pMON38755, with the following exception. The 0.15 kb psbApromoter sequence is replaced with the Prrn/G10L promoter sequencedescribed above.

1H. Constructs for the Expression of Aprotinin in Plastids

A series of constructs were prepared to direct the expression of thepharmaceutical protein aprotinin from the plastid. The nucleic acidsequence encoding for aprotinin (FIG. 2) was cloned into a plastidexpression construct to control the expression of aprotinin from the T7gene 10 leader promoter which is induced from a nuclearly expressed,plastid targeted T7 Polymerase. The constructs used in which theaprotinin sequence was cloned are as described in U.S. Pat. No.5,576,198, the entirety of which is incorporated herein by reference.The plastid transformation vector pCGN6146 is designed by replacing theDNA sequence encoding for GUS from pCGN4276 (described in U.S. Pat. No.5,576,198) with the coding sequence of aprotinin. The tobacco plastidtransformation construct pCGN6147 contains the same elements as pCGN6146except pCGN6147 contains the six 5′ amino acids of the GUS encodingsequence ligated to the 5′ terminus of the aprotinin encoding sequence.The six amino acids of the 5′ terminus of the GUS nucleotide sequenceare included to aid in the translation of the aprotinin protein. Thetobacco plastid transformation vector pCGN6156 is essentially the sameas pCGN4276 except the coding region of aprotinin is cloned to the 3′end of the GUS coding sequence. Thus, pCGN6156 contains as operablylinked the T7 promoter, a DNA sequence encoding for GUS fused with theDNA sequence encoding for aprotinin and the psbA 3′ transcriptiontermination sequence.

A plastid expression construct, pCGN6154, was constructed from pCGN4276by replacing the GUS coding sequence with the aprotinin protein operablylinked to the 3′ terminus of the coding sequence of cytochrome f (petA)of the tobacco chloroplast. Thus, pCGN6154 contains the T7 promotersequence operably linked to the nucleotide sequence of petA andaprotinin. The petA sequence is included to direct the expressedaprotinin protein to the thylakoid.

Example 2 Plant Transformation

2A. Nuclear Transformation

Tobacco plants transformed to express the constructs pWRG4744 andpWRG4747 in the nucleus of a plant cell may be obtained as desribed byHorsch et al. (Science (1985) 227:1229-1232).

2B. Plastid Transformation

Tobacco plastids are transformed by particle gun delivery ofmicroprojectiles as described by Svab and Maliga (Proc. Natl. Acad. Sci.(1993) 90:913-917), and described here.

Dark green, round leaves are cut, preferably from the middle of theshoots, from 3-6 week old Nicotiana tabacum cv. Havana which have beenmaintained in vitro on hormone free MS medium (Murashige and Skoog,(1962) Physiol Plant. 15, 473-497) supplemented with B5 vitamins inPhytatrays or sundae cups with a 16 hour photoperiod at 24° C. Each cutleaf is then placed adaxial side up on sterile filter paper over tobaccoshoot regeneration medium (TSO medium: MS salts, 1 mg/lN⁶-benzyladenine, 0.1 mg/l 1-naphthaleneacetic acid, 1 mg/l thiamine,100 mg/l inositol, 7 g/l agar pH 5.8 and 30 g/l sucrose). Leaves arepreferably placed in the center of the plate with as much contact withthe medium as possible. The plates are preferably prepared immediatelyprior to use, but may be prepared up to a day before transformation byparticle bombardment by wrapping in plastic bags and storing at 24° C.overnight.

Tungsten or gold particles are sterilized for use as microcarriers inbombardment experiments. Particles (50 mg) are sterilized with 1 ml of100% ethanol, and stored at −20° C. or −80° C. Immediately prior to use,particles are sedimented by centrifugation, washed with 2 to 3 washes of1 ml sterile deionised distilled water, vortexed and centrifuged betweeneach wash. Washed particles are resuspended in 500 μl 50% glycerol.

Sterilized particles are coated with DNA for transformation. Twenty-fivemicoliter aliquots of sterilized particles are added to a 1.5 mlmicrofuge tube, and 5 μg of DNA of interest is added and mix by tapping.Thirty-five microliters of a freshly prepared solution of 1.8M CaCl₂ and30 mM spermidine is added to the particle/DNA mixture, mixed gently, andincubated at room temperature for 20 minutes. The coated particles aresedimented by centrifuging briefly. The particles are washed twice byadding 200 μl 70% ethanol, mixing gently, and centifuging briefly. Thecoated particles are resuspended in 50 μl of 100% ethanol and mixedgently. Five to ten microliters of coated particles are used for eachbombardment.

Transformation by particle bombardment is carried out using the PDS 1000Helium gun (Bio Rad, Richmond, Calif.) using a modified protocoldescribed by the manufacturer.

Plates containing the leaf samples are placed on the second shelf fromthe bottom of the vacuum chamber and bombarded using the 1100 p.s.i.rupture disk. After bombardment, petriplates containing the leaf samplesare wrapped in plastic bags and incubated at 24° C. for 48 hours.

After incubation, bombarded leaves are cut into approximately 0.5 cm²pieces and placed abaxial side up on TSO medium supplemented with 500μg/ml spectinomycin. After 3 to 4 weeks on the selection medium, small,green spectinomycin resistant shoots will appear on the leaf tissue.These shoots will continue to grow on spectinomycin containing mediumand are referred to as primary putative transformants.

When the primary putative transformants have developed 2 to 3 leaves, 2small pieces (approximately 0.5 cm²) are cut from each leaf and used foreither selection or for a second round of shoot regeneration. One pieceis placed abaxial side up on plates containing TSO medium supplementedwith 500 μg/ml spectinomycin, and the other piece is placed abaxial sideup on TSO medium supplemented with 500 μg/ml each of spectinomycin andstreptomycin. Positive transformants are identified as the shoots whichform green callus on the TSO medium containing spectinomycin andstreptomycin.

After 3 to 4 weeks, the tissue placed on TSO medium containing onlyspectinomycin, which has been identified as positive on the TSO mediumwith spectinomycin and streptomycin, will develop green shoots. Two tofour shoots of each positive transformant are selected and transferredto TSO medium supplemented with 500 μg/ml spectinomycin for generationof roots. Southern analysis is performed on 2 shoots to confirmhomoplasmy as described below. Shoots from homoplasmic events aretransferred to the greenhouse for seed production, while transformantswhich are not homoplasmic are sent through a second round orregeneration on TSO medium with 500 μg/ml spectinomycin to attainhomoplasmy.

Example 3 Analysis of Transplastomic Tobacco Plants Transformed withHerbicide Tolerance Constructs

3A. Southern Analysis

Transformed plants selected for marker aadA marker gene expression areanalyzed to determine whether the entire plastid content of the planthas been transformed (homoplastic transformants). Typically, followingtwo rounds of shoot formation and spectinomycin selection, approximately50% of the transgenic plantlets which are analyzed are homoplastic, asdetermined by Southern blot analysis of plastid DNA. Homoplasmicplantlets are selected for further cultivation.

Genomic DNA is isolated from transformed tobacco plants,electrophoresed, and transferred to filters as described in Svab et al.((1993), Proc Natl Acad Sci, 90:913-917).

Homoplasmic tobacco plants transformed to express CP4 EPSPS in plastidswere identified using a probe prepared from a 2.4 kb EcoRI/EcoRVfragment from the vector pOVZ2 (similar to pOVZ15 described in Zoubenko,et al. 1994, supra). The 2.4 kb probe fragment encompasses part of thetargeting sequence.

Results of the Southern hybridizations identified 3 homoplasmic linesfrom tobacco transformed with the constructs pMON30123 and pMON30130 and1 line from tobacco transformed with pMON38773 for further analysis.

The complete disappearance of the 3.27 Kb native tobacco BamHI fragmentin the lines 30123-19-1A, 30123-23-2A, 30123-18-1B, 30130-51-2A,30130-51-2P, 30130-51-1P, and 38773-6 with a probe covering the regionof integration, and the appearance of expected sized bands for theinserted DNA fragments in those transformants, 5.14 kb and 0.9 kb,establishes that the transformed plants are homoplasmic for the intendedconstructs.

Results of the Southern hybridizations identified 3 homoplasmic linesfrom tobacco transformed with pCGN5177, lines 74-1B-P, 74-2 and 74-7.

Transplastomic 5175 and 6114 tobacco lines were analyzed by Sourthernhybridization for homoplasmy as described above. Results of the Southernhybridizations identified 4 homoplasmic lines from tobacco transformedwith pCGN6114.

Results from hybridizations of 5175 transplastomic tobacco linesidentified one line, 76-4A-F, as homoplasmic, and a second line as 95%homoplasmic.

Homoplasmic tobacco plants transformed to express BXN/AHAS in plastidswere identified using Southern hybridizations as described above.

Results of the Southern hybridizations identified 14 homoplasmic linesfrom tobacco transformed with pCGN5026. The filters were reprobed with aBXN gene fragment, and 21 lines were found to contain BXN, 14 lines ofwhich were homoplasmic.

3B. Northern Analysis

In order to determine the level of transcription of the EPSPS, BXN orAHAS mRNA expressed in the transplastomic tobacco plants, Northern blothybridizations were performed with total RNA isolated from each of thelines identified. Total RNA was isolated using TRIzol reagent (Gibco-BRLLife Technologies, Gaithersburg, Md.) according to the manufacturersprotocol. Total RNA, 2 μg, was separated on a denaturing agarose gel andtransferred to nylon membrane (Maniatis et al., 1989, supra).Radioactive probes for hybridizations were prepared using random primerlabeled (using Random Primer labeling kit from Boehringer Mannheim) CP4EPSPS, phytoene desaturase, BXN, or AHAS fragments and hybridizationswere carried out in 2×SSPE (Maniatis, et al., 1989, supra), at 60° C.Filters were stripped and reprobed with a plastid 16S ribosomal RNA geneprobe (from pPRV112A, Zoubenko, et al., 1994, supra) to confirmhomogenous loading of RNA on the filter.

Results of the Northern hybridizations performed with EPSPS probesdemonstrate that all seven (7) lines examined express CP4 EPSPS mRNA.Hybridizations performed with the 16S ribosome probe confirm thatdenaturing gels were loaded with similar amounts of total RNA for eachsample. Furthermore, transplastomic tobacco lines expressing EPSPS fromthe Prrn/rbcL(RBS) (pMON30123) regulatory elements express EPSPS mRNA tohigher levels than tobacco plants homoplasmic for EPSPS controlled bythe Prrn/G10L (pMON38773) promoter/RBS sequences.

Results of Northern hybridizations performed with BXN, AHAS and crtIprobes demonstrates that all homoplasmic 5026, 5175, and 5177 tobaccolines expressed crtI, BXN and/or AHAS mRNA.

3C. Western Blot Analysis of Tobacco CP4 EPSPS

To determine the expression of the EPSPS Western blot analysis wasperformed on a single line from each construct, pMON30123, pMON30130,and pMON38773.

Total soluble protein was extracted from frozen leaf tissue by grinding250 mg tissue in 250 μl of PBS buffer (1 mM KH₂PO₄, Na₂HPO₄, 0.137MNaCl, 2.7 mM KCl pH 7.0) containing protease inhibitors. The homogenateis centrifuged for 5 minutes, and the supernatant is transferred to afresh tube. The concentration of the protein in the supernatant isdetermined using a protein concentration assay (BioRad, Richmond,Calif.).

Extracted total protein is electrophoresed on a 4-20% SDS-PAGE gel(Sigma, St Louis, Mo.), and transferred to PVDF membrane in 1×SDS-PAGEbuffer (Maniatis et al. 1989, Cold Spring Harbor Press). Standards ofquantitated purified CP4 EPSPS protein were used to quantify theexpression of the CP4 EPSPS as expressed in the plant plastid.

Western hybridizations are performed as described in Staub and Maliga(1993) EMBO Journal, 12(2) 601-606, except using antibodies raised toEPSPS. PVDF membranes containing the transferred electrophoresed proteinwere incubated in a blocking solution of PBS buffer containing 0.05%Tween-20 (PBS-T) and 5% milk overnight at 4° C. The membranes are thenincubated in a solution of PBS-T containing 1% milk and a primaryantibody raised in goats to the CP4 EPSPS for 2 hours at roomtemperature. The membranes are washed three times in a solution of PBS-Tcontaining 0.1% milk, each wash for 5 minutes at room temperature. Themembranes are then incubated in a solution of PBS-T containing 1% milkand sheep anti-goat antibody for 1 hour at room temperature, and washedagain in PBS-T containing 0.1% milk, three times for 10 minutes at roomtemperature. A final wash using only PBS-T is performed beforedeveloping the membranes using anonradioactive detection kit (ECL,Amersham).

TABLE 2 Construct Number % Total Soluble Protein pMON30123 0.001pMON30130 0.002 pMON38773 0.2 pMON38798 0.2 pMON45259 >12.0 pMON49218>12.0

The results listed in Table 2 demonstrate that significant increases inthe level of EPSPS protein may be obtained from plants transformed toexpress EPSPS from the Prrn/G10L promoter. These results demonstratethat EPSPS expression driven by the Prrn/rbcLRBS regulatory sequencesmay produce approximately 0.001% of the total soluble protein as EPSPS,while in plants expressing EPSPS from the Prrn/G10L regulatory sequencesexpress 0.2% of the total soluble protein as EPSPS. Subsequent lineshave demonstrated total soluble protein of about 1% EPSPS when expressedfrom the Prrn/G10L regulatory sequences. These results, taken togetherwith the results of the Northern hybridizations above, indicate thatmore efficient translation may be obtained from the G10L ribosomebinding site.

Furthermore, plastid expression constructs containing the N-terminal 14amino acid from GFP demonstrated high levels of protein expression.Transplastomic lines containing either pMON45259 or pMON49218demonstrated total soluble protein of greater than 12% CP4 EPSPS.

Western immunoblot hybridization were also performed on 2 homoplasmic5026 tobacco lines as described above, using antibodies raised againstbromoxynil. The results of Western immunoblot analysis of total solubleprotein extracted from tobacco lines transformed with pCGN5026demonstrated that both homoplasmic lines produced nitrilase protein.

Western immunoblot analysis was performed as described above from totalprotein extracted from tobacco lines transformed with pCGN6114 andpCGN5197.

The results of the analysis demonstrated that bromoxynil was produced in6114 tobacco lines ranging from 1% to 2% of the total soluble leafprotein.

The results of the Western analysis of the 20 5197 tobacco linesdemonstrated that bromoxynil and Bt were both produced as 1% of thetotal soluble leaf protein.

3D. Analysis of EPSPS Enzyme Activity

The EPSPS enzyme activity in transplastomic tobacco plants containingthe plastid expression vector pMON38773 was determined using a highpressure liquid chromatography (HPLC) assay.

Methods for the analysis of EPSPS enzyme activity are described inPadgette et al. (J. Biol. Chem. (1988)263:1798-1802 and Arch. Biochem.Biophys. (1987)258:564-573) and Wibbenmeyer et al. (Biochem. Biophys.Res. Commun. (1988)153:760-766). The results are summarized in Table 3below.

TABLE 3 Nuclear Enzymatic Nuclear Activity % Total Plants ChloroplastRange In Range 38773-6 1-3.7 μmol/mg 1% >0.1 μmol/mg 16% >10 nmol/mg 55%16.39 nmol/mg >1 nmol/mg 32% 0 nmol/mg 3%

These results demonstrate that EPSPS expression in plastids producesactive EPSPS enzyme.

3E. Analysis for Glyphosate Tolerance

A transplastomic tobacco line homoplasmic for the construct pMON38773was tested in vitro to determine the highest level of glyphosatetolerance. Explant tissue was prepared from leaf pieces of nontransgenicwild type tobacco control, Havanna, plants and the homoplasmic tobaccoline 38773-6 and cultured for regeneration of shoots on TSO medium(described above) supplemented with glyphosate levels of 50 μM, 75 μM,100 μM, 150 μM and 200 μM. The results are summarized in Table 4 below.The number of explants producing shoots was determined at 3 weeks and 6weeks after explant preparation and culturing on glyphosate containingmedium.

TABLE 4 Total Number Number Glyphosate Number Regenerating Regenerating% Explant Level (μM) Explants 3 Weeks 6 Weeks Regeneration Wild Type 5010 0 0 0 75 10 0 0 0 100 10 0 0 0 150 10 0 0 0 200 10 0 0 0 38773-6 50 85 8 100 75 18 14 18 100 100 17 12 15 88 150 18 10 16 89 200 16 8 15 86

The above results demonstrate that at all levels of glyphosate examined,shoots regenerated from explants prepared from a tobacco linehomoplasmic for pMON38773, while no shoots regenerated from explantsprepared from nontransformed control plants. These results suggest thattobacco plants expressing EPSPS in plastids demonstrate tolerance toglyphosate levels of at least 200 μM.

Additional transplastomic lines were tested in vitro for glyphosatetolerance as bed above. The results are shown in Table 5.

TABLE 5 Summary of tobacco plastid transformation experiments withvarious constructs containing EPSPS genes. No. of shoots ConstructSpec/strep (+) Gly 50 uM(+) pMON38766 (Wild) 1 0 pMON38766 (T7) 6 0pMON38773 (Wild) 9 5 (1) pMON38797 (Wild) 2 0 pMON38798 6 6 pMON38793 80 pMON38796 4 0 pMON45201 9 3 pMON45204 12 * (No. of shoots positive at1 mM glyphosate)

These results demonstrate that these transplastomic lines show toleranceto glyphosate. The numbers in parentheses are the number of shootsresistant to selection at 1 mM glyphosate. Thus, as can be seen in table5, tobacco lines are generated that are tolerant of selection at 1 mMglyphosate.

Homoplasmic tobacco plants of the line 38773-6 are sprayed withglyphosate using a track sprayer at concentrations corresponding to 0oz/acre, 16 oz/acre, 32 oz/acre and 64 oz/acre to test for whole planttolerance. Plant height was measured before and after s praying withglyphosate. The vegetative injury data was collected two weeks afterspraying, while the reproductive injury data was collected at plantmaturity.

Initial results indicate that homoplasmic tobacco lines sprayed aretolerant of glyphosate at the concentration of 16 oz/acre asdemonstrated in the vegetative tissue injury (Table 6). As can be seenin Table 5 transplastomic lines were generated which demonstrated a goodlevel of glyphosate tolerance at 32 oz/acre. In subsequent experimentswith additional transformed lines, transplastomic lines have showntolerance to glyphosate at a level of 64 oz/acre.

Tolerance is characterized by the continued growth and greening oftissues sprayed with glyphosate. However, as the concentration ofglyphosate applied increased, there was a corresponding increase in thelevel of vegetative injury. In contrast, nontransformed control plantswhich were highly susceptible to glyphosate concentrations as low as 16oz/acre.

TABLE 6 Round- up Plant height Plant height Vegeta- Fer- Plant rate (cm)before (cm) after tive tility No. Construct (oz/A) spray spray injuryrating 1 38773 0 12.2 30.5 0 0 2 38773 0 13.6 34.0 0 0 3 38773 0 8.623.8 0 0 4 38773 0 8.6 26.2 0 0 5 38773 0 7.8 28.8 0 0 6 38773 0 12.831.5 0 0 7 38773 0 12.2 31.6 0 0 8 38773 0 11.6 35.5 0 0 9 38773 16 9.029.0 1 0 10 38773 16 14.4 31.0 0 0 11 38773 16 13.4 32.0 0 0 12 38773 1613.2 30.0 0 0 13 38773 16 14.2 30.5 0 1 14 38773 16 14.0 33.0 0 0 1538773 16 13.2 30.2 0 0 16 38773 16 14.9 30.4 0 0 17 38773 32 12.0 26.5 24 18 38773 32 11.6 25.4 1 1 19 38773 32 9.4 22.0 1 3 20 38773 32 11.223.0 2 4 21 38773 32 13.8 25.8 1 2 22 38773 32 12.4 23.0 1 4 23 38773 3210.2 19.0 2 4 24 38773 32 13.8 23.2 2 3 26 38773 64 11.8 20.0 2 5 2738773 64 13.0 22.0 2 5 28 38773 64 12.2 18.0 3 5 29 38773 64 15.8 23.0 25 30 38773 64 10.4 17.5 2 5 32 38773 64 15.0 18.5 2 5 33 38773 64 13.821.8 2 5 34 38773 64 13.6 19.0 3 5 35 38773 64 10.8 16.0 3 5 36 Wildtype 0 21.0 40.6 0 0 37 Wild type 0 16.0 38.0 0 0 38 Wild type 0 15.034.6 0 0 39 Wild type 0 17.6 32.2 0 0 40 Wild type 0 15.0 31.6 0 0 41Wild type 0 14.0 32.0 0 0 42 Wild type 16 10.0 11.8 3 5 43 Wild type 168.0 10.0 3 5 44 Wild type 16 8.6 11.0 3 5 45 Wild type 16 8.0 14.0 3 546 Wild type 16 9.8 11.0 3 5 47 Wild type 16 10.4 14.0 3 5 48 Wild type32 10.8 13.2 3 5 49 Wiid type 32 9.0 13.0 3 5 50 Wild type 32 8.0 10.2 35 51 Wild type 32 11.0 14.0 4 5 52 Wild type 32 9.8 13.0 3 5 53 Wildtype 32 8.0 10.8 4 5 54 Wild type 64 7.5 8.6 4 5 55 Wild type 64 11.212.5 4 5 56 Wild type 64 10.2 12.8 4 5 57 Wild type 64 11.5 13.0 4 5 58Wild type 64 13.0 15.0 4 5 59 Wild type 64 9.8 11.2 4 5 Vegetativeinjuries: 0 = normal plant 1 = slight chlorosis of new leaves andstunting 2 = severe chlorosis of new leaves, malformation of new leaves,and severe stunting 3 = dying plant 4 = dead plant Fertility ratings: 0= Fertile, no delay in maturity, lots of seed 1 = Some abortion, slightdelay in seed set, seed 2 = Significant abortion, significant delay inseed set, some seed 3 = Very severe abortion, immature seed pots, a fewseed 4 = malformed flowers; if flowered, extreme delay in flowering andno seed produced 5 = dead plant

In addition, other transplastomic lines were analyzed for tolerance tospraying with various levels of glyphosate as described above. Specificactivity is measured as the amount of exogenously added Phosphoenolpyruvate (PEP) converted to Shikimate-3-phosphate (S3P) per unit proteinin the plant extract. Addition of glyphosate tests sensitivity of theEPSPS enzyme to glyphosate. The results are summarized in Table 7.

TABLE 7 SPECIFIC SPECIFIC % TOTAL ACTIVITY ACTIVITY VegetativeReproductive SOLUBLE (nmol/min/mg) (nmol/min/mg) tolerance toleranceLINE PROTEIN No gly +1 mM gly (oz/acre) (oz/acre) Wild-type 3.4 0 0 0pMON10154 0.04 19.0 18.4 128 64 pMON45201 — 301.6 221.2 32 32 pMON45204— 339.9 371.8 128 64 pMON30123 0.001 4.0 0 0 0 pMON30130 0.002 6.2 0 0 0pMON38773 0.2 16.7 6.7 32 16 pMON38798 0.2 17.2 14.7 32 16pMON45259 >12.0 — — 128 64 pMON49218 >12.0 — — 128 64

These data demonstrate that high levels of glyphosate tolerance can beobtained in transplastomic plants expressing various EPSPS sequences. Inparticular, lines pMON45204, pMON45259, and pMON49218 provide toleranceto glyphosate applied at levels of at least 128 oz/acre on vegetativetissues, and at least 64 oz/acre on reproductive tissues.

Furthermore, constructs pMON42259 and pMON49218 provide for high levelexpression of CP4 EPSPS from plant plastids transformed with theseconstructs. In particular, expression levels of greater than about 12percent total soluble protein are obtained in constructs employingsequences encoding the first 14 amino acids of GFP fused to theN-terminus of CP4.

3F. BT/BXN Analysis

Homoplasmic tobacco plants of the lines 5175 and 5197 are sprayed withBuctril herbicide at a concentration of 4% to test for whole planttolerance.

Results of the spray test with Buctril demonstrated that all 5197 linesexpressing bxn were completely resistant when sprayed with a solutioncontaining 4% Buctril herbicide.

Two lines out of six 5175 lines tested were completely resistant to theherbicide when sprayed with a 4% solution containing Buctril.

3G. Norflurazon Resistance Analysis

An experiment was set up to determine the efficacy of the Crt I traitwith respect to resistance to the herbicide Norflurazon. Three 5177transformed lines, 74-1B-P, 74-2-A, and 74-7-C and three control lineswere planted. Plants were grown for seven weeks and then watered with a3 μM Norflurazon solution. Plants negative for the presence of the crtIplastid-borne gene were bleached by Norflurazon treatment, positiveplants stayed green and continued to grow.

The results show that the three homoplasmic 5177 tobacco lines wereresistant to the 3 μM Norflurazon solution, while the control plantswere all susceptible to the solution (Table 8).

TABLE 8 Line Control/Transgenic Result Xanthi Control Susceptible 2560AXanthi Control Susceptible 75-5D-A Control Susceptible 74-1B-Phomoplasmic Resistant 74-2-A homoplasmic Resistant 74-7-C homoplasmicResistant

Example 4 Analysis of hGH Transgenic Tobacco Plants

4A. Southern Analysis

Transformed plants selected for aadA marker gene expression are analyzedto determine whether the entire plastid content of the plant has beentransformed (homoplastic transformants). Homplasmic plants are selectedusing Southern hybridization for further cultivation.

Genomic DNA is isolated from transformed tobacco plants,electrophoresed, and transferred to filters as described in Svab et al.((1993), Proc Natl Acad Sci, 90:913-917). Homoplasmic tobacco plantstransformed to express hGH were identified using a probe prepared from a2.4 kb EcoRI/EcoRV fragment from the vector pOVZ2 (similar to pOVZ15described in Zoubenko, et al. 1994, supra). The 2.4 kb probe fragmentencompasses part of the targeting sequence.

The complete disappearance of the 3.27 Kb native tobacco BamHI fragmentin the lines with a probe covering the region of integration, and theappearance of the expected size band for the inserted DNA fragments inthose transformants, 5.6 kb, establishes that the transformed plants arehomoplasmic for the intended constructs.

4B. Protein Expression Analysis

Homoplasmic tobacco lines expressing hGH and nuclear tobaccotransformants are used to determine the expression of the hGH protein.Western blot analysis was performed on tobacco lines containingconstructs pWRG4838, pMON38755 and pMON38794 for plastid expression andan ELISA assay was used for transgenic tobacco lines containing pWRG4744and pWRG4747 for nuclear expression of hGH.

Total protein extractions and western blot procedures were performed asdescribed above, with the exception of the primary antibody was raisedagainst hGH.

TABLE 9 Expression Levels of hGH in Tobacco Nuclear Genome and Plastidgenome Expression Level Construct Expression % Total Soluble ProteinpWRG4744 nuclear 0.002-0.125% pWRG4747 nuclear 0.002-0.025% pWRG4838plastid 0.2% pMON38755 plastid 1.0% pMON38794 plastid 7.0%

Results of the Western analysis (Table 9) demonstrates that hGHexpressed in plastids of plant cells accumulates to significantly higherlevels than hGH expressed in the nucleus and targeted to either thecytoplasm or plastid of plant cells. Tobacco plants transformed toexpress hGH in the nucleus accumulated hGH levels of 0.002% (cytoplasmictargeted) to 0.025% (plastid targeted) of total soluble leaf protein,while tobacco plants expressing hGH in the plastid accumulated hGHlevels of 0.2% to 7.0% of the total soluble leaf protein as hGH.Furthermore, homoplasmic tobacco plants expressing hGH directed from thePrrn/G10L regulatory sequences accumulate 35 fold higher levels of hGHthan homoplasmic tobacco plants expressing hGH directed from the PpsbApromoter sequence.

4C. Characterization of hGH Protein Expressed in the Plastid

In order to determine whether the hGH expressed from plastids wasproperly processed, experiments were performed to determine correctfolding and bioacitivity.

Two bottom leaves of transplastomic tobacco lines containing pMON38794were used to extract and purify hGH. Large veins were removed from theexcised leaves, and the leaf tissue was cut into small sections(approximately 0.5 cm²). The leaf pieces were flash frozen in liquidnitrogen and ground to a fine powder in a chilled mortar and pestle. Tengrams of frozen, ground leaf tissue was added to ice cold 100 mM Trisbase solution (30 ml) and mixed vigorously by vortexing for 5 minutes.The solution was filtered through a single layer of cheese cloth.

From the filtered solution, three separate samples were prepared. Thefirst sample was prepared by cetrifuging 4 ml of the filtrate for 1minute at 16,000 rpm. The centrifugate was aliquoted into 1 ml vials andfrozen in dry ice. The remaining filtrate was centrifuged for 10 minutesat 4800 rpm, and several 0.5 ml aliquots were frozen as above for thesecond sample.

To the remaining centrifuged filtrate (approximately 25 ml), 200 μl ofglacial acetic acid was added to lower the pH from 8.2 to 4.56. Thesolution was centrifuged at 4800 rpm for 30 minutes, and the supernatantwas frozen over dry ice for the third sample.

Total soluble protein (TSP, Table 10) was calculated in these samples bystandard protein assay procedures (Maniatis,), and the percent purity ofhGH was calculated based on results from Western blot analysis usingknown concentrations of starting material.

TABLE 10 TSP GP2000 Sample ID mg/mL mg/L % Purity Filtered Extractimmediately centrifuged 6.3 28 0.45% and frozen Filtered extractcentrifuged at 4800 rpm 6.4 28 0.45 for 10 mm and frozen pH adjusted andcentrifuged extract 0.75 21 2.8%

The pH adjusted and centrifuged extract was purified by ReversePhase-HPLC (RP-HPLC) for electrospray mass spectrometry andamino-terminal amino acid sequencing. RP-HPLC was performed using aPerkin-Elmer series 200 pump and autosampler and a Vydac C8 (250 by 4.6mm) RP-HPLC column. 750 microliters of sample was loaded onto the columnequilibrated with 20 mM trifluoroacetic acid (TFA) and 50% acetonitrile.After loading, the column was washed for 2 minutes with 50%acetonitrile, 20 mM TFA followed by a 2% linear acetonitrile gradientover 10 minutes followed by a 10% acetonitrile gradient over 1 minute.The flow rate was a constant 1.5 ml/minute with the column eluatemonitored at 278 nm with a Perkin-Elmer 785 detector. Data was collectedand analyzed with a PE-Nelson Turbochrom data system.

The results of the RP-HPLC analysis are shown in FIG. 3. Peak I (tallestpeak) has the retention time expected for properly folded, native 22 kDaGP2000. This peak was collected and dried down in a Savant Speed-Vac foramino terminal sequencing and electrospray mass spectrometry.

Electrospray ionization mass spectrometry (MS) analysis used a MicromassQ-T of electrospray time-of-flight mass spectrometer. The samples wereprepared byresuspending in 50% methanol+2% acetic acid, and infused intothe source of the mass spectrometer at a rate of 4 mL/min. The raw datashown in FIG. 4 shows a series of ions corresponding to the specie(s)present in the sample with varying numbers of protons attached. The axesof this spectrum are intensity versus mass-to-charge ratio of thespecie(s) present. A deconvolution algorithm is used to convert thisseries of multiply charged ions into a molecular weight spectrum.

The results of the mass spectrometry of the RP-HPLC peak I shows 4 majorprotein species of different molecular mass. The 21,997 kDa speciesrepresents the predicted mass of hGH with the predicted N-terminal Pheremoved by over-cleavage of the Ubiquitin protease. The 22,124 kDAspecies represents the predicted mass of properly processed, correctamino acid sequence of hGH. The 22,507 kDA and 22,664 kDA species arethought to represent an hGH with the N-terminal Phe and hGH which havebeen modified during plant extraction procedures, respectively. Thecalculated molecular mass of the proteins suggests that the hGHexpressed from the plastid is properly folded (i.e. the correctdisulfide bonds are created).

Amino terminal sequencing was done by standard Edman degradation, andconfirmed the N-terminal sequences discussed above.

4D. Bioactivity of hGH Expressed in Plant Plastids

Bioactivity of the pH adjusted and centrifuged extract was tested usingcells from an Nb2 cell line. These cells proliferate in the presence ofgrowth hormone and other estrogenic type compounds. The assay involvesputting various concentrations of growth hormone-containing extract intoa 96 well plate. Then a constant amount of cells are added to each well.The plate is incubated for 48 hrs and then a reagent called MTS isadded. Metabolizing cells take up the MTS and convert it to a bluecolored substance. The more cells there are the more blue color in thewell. The blue color is measured using a spectrophotometer. The numberof cells should be proportional to the concentration of growth hormonein the media. At some high concentration one expects that the cells willbecome saturated with growth hormone and that the dose response willlevel off. At very low hGH concentrations essentially no enhanced growthis seen. A sigmoidal shape grapf is expected to be produced graphing thecell number (or absorbance) vs hGH concentration graph.

The results of the bioactivity assay (FIG. 5) demonstrates that the hGHexpressed from a plant plastid has a sigmoidal shape when graphed asabsorbance vs hGH concentratioin.

Example 5 Analysis of Aprotinin Transplastomic Tobacco Plants

5A. Western Analysis of Aprotinin Expression in Plastids

Homoplasmic tobacco lines expressing are used to determine theexpression of the aprotinin protein. Western blot analysis was performedon tobacco lines containing constructs pCGN6146, pCGN6147, pCGN6154 andpCGN6156 for plastid expression of aprotinin.

Total protein extractions and western blot procedures were performed asdescribed above, with the exception of the primary antibody was raisedagainst aprotinin.

The results of the Western analysis indicate that aprotinin is expressedfrom the T7 polymerase promoter when the aprotinin coding sequence isfused with either the PetA or full length GUS gene. Furthermore, theseresults indicate that the petA sequence efficiently targets theaprotinin protein to the plant cell thylakoid.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

7 1 44 DNA Artificial Sequence Synthetic Oligonucleotide 1 aattgtagaaataattttgt ttaactttaa gaaggagata tacc 44 2 44 DNA Artificial SequenceSynthetic Oligonucleotide 2 ggtatatctc cttcttaaag ttaaacaaaa ttatttctacaatt 44 3 44 DNA Artificial Sequence Synthetic Oligonucleotide 3catgggtata tctccttctt aaagttaaac aaaattattt ctac 44 4 58 PRT Humanaprotinin 4 Arg Pro Asn Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys LysAla 1 5 10 15 Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu CysGln Thr 20 25 30 Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe LysSer Ala 35 40 45 Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55 5 168 DNAArtificial Sequence Synthetic Oligonucleotide 5 gaattcgagc tcggtacccaaagctccccc gccgtcgttc aatgagaatg gataagaggc 60 tcgtgggatt gacgtgagggggcagggatg gctatatttc tgggagcgaa ctccgggcga 120 attgtagaaa taattttgtttaactttaag aaggagatat acccatgg 168 6 244 DNA Artificial SequenceSynthetic Oligonucleotide 6 gaattcggta cccccgtcgt tcaatgagaa tggataagaggctcgtggga ttgacgtgag 60 ggggcaggga tggctatatt tctgggagcg aactccgggcgaatactgaa gcgcttggat 120 acaagttatc cttggaagga aagacaattc cggatcctctagaaataatt ttgtttaact 180 ttaagaagga gatataccca tgggtaaagg agaagaacttttcactggag ttgtcccaag 240 catg 244 7 15 PRT Artificial Sequence PeptideSequence 7 Met Gly Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ser Met 15 10 15

What is claimed is:
 1. A construct comprising the following componentsin the 5′ to 3′ direction of transcription: a) a promoter functional ina plant plastid and a ribosome binding site operably joined thereto,said ribosome binding site selected from a T7 bacteriophage gene 10leader or a rbcL RBS; b) a DNA sequence encoding a protein which iscapable of conferring tolerance in a plant cell to at least oneherbicide compound when said DNA sequence is transcribed in plastids ofsaid plant cell; and c) a transcription termination region.
 2. Theconstruct according to claim 1 wherein said DNA sequence encodes a geneproduct which confers tolerance to the herbicide glyphosate.
 3. Theconstruct according to claim 2 wherein said DNA sequence encodes aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase.
 4. Theconstruct according to claim 3 wherein said DNA sequence is selectedfrom the group consisting of the E. coli or Salmonella aroA gene, theAgrobacterium CP4 gene, mutant petunia EPSPS gene, mutant EPSPS gene ofPsuedomonas strain LBAA, and the Bacillus subtilis aroE gene.
 5. Theconstruct according to claim 2 wherein said DNA sequence encodes aglyphosate-modifying enzyme.
 6. The construct according to claim 5wherein said glyphosate-modifying enzyme is encoded by a gene selectedfrom the group consisting of the gox, hph, glpA and glpB genes.
 7. Theconstruct according to claim 1 wherein said DNA sequence is unmodifiedwith respect to the native DNA sequence encoding said protein.
 8. Theconstruct according to claim 1 wherein said DNA sequence is syntheticwith respect to the native DNA sequence encoding said protein.
 9. Theconstruct according to claim 1 wherein said DNA sequence encodes asulphonylurea-tolerant AHAS.
 10. The construct according to claim 1wherein said DNA sequence encodes a imidizalinone-tolerant AHAS.
 11. Theconstruct according to claim 1 wherein said DNA sequence encodes aphosphinothricin-tolerant gene product.
 12. The construct according toclaim 11 wherein said DNA sequence is the BAR gene.
 13. The constructaccording to claim 1 wherein said DNA sequence encodes an enzyme of thecarotenoid pathway.
 14. The construct according to claim 13 wherein saidDNA sequence is the crtI gene.
 15. The construct according to claim 1wherein said DNA sequence encodes a bromoxynil-tolerant gene product.16. The construct according to claim 15 wherein said bromoxynil-tolerantgene is the bxn gene.
 17. A plant cell plastid containing the constructaccording to claim
 1. 18. A plant, plant seed, plant cell or progenythereof each containing a plant plastid according to claim
 17. 19. Amethod for producing tolerance of a herbicide in a plant cell, whereinsaid method comprises transforming plastids of said plant cell with aconstruct comprising the following as operably joined components in the5′ to 3′ direction of transcription: a) a promoter functional in a plantplastid and a ribosome binding site operably joined thereto, saidribosome binding site selected from a T7 bacteriophage gene 10 leader ora rbcL RBS; b) a DNA sequence encoding a protein which is capable ofconferring in a plant cell tolerance to at least one herbicide compoundwhen said DNA sequence is transcribed in plastids of said plant cell;and c) a transcription termination region, and growing plant cellscomprising said transformed plastids under conditions wherein said DNAsequence is transcribed whereby plant cells containing said plantplastids are rendered tolerant to applications of said at least oneherbicide compound.
 20. The method according to claim 19 wherein saidDNA sequence encodes a gene product which confers tolerance to theherbicide glyphosate.
 21. The method according to claim 20 wherein saidDNA sequence encodes a glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate synthase.
 22. The method according toclaim 21 wherein said DNA sequence is selected from the group consistingof the E. coli or Salmonella aroA gene, the Agrobacterium CP4 gene,mutant petunia EPSPS, mutant EPSPS gene of Psuedomonas strain LBAA andthe Bacillus subtilis aroE gene.
 23. The method according to claim 20wherein said DNA sequence encodes a glyphosate-modifying enzyme.
 24. Themethod according to claim 23 wherein said glyphosate-modifying enzyme isencoded by a gene selected from the group consisting of the gox, hph,glpA and glpB genes.
 25. The method according to claim 19 wherein saidDNA sequence is unmodified with respect to the native DNA sequenceencoding said protein.
 26. The method according to claim 19 wherein saidDNA sequence is synthetic with respect to the native DNA sequenceencoding said protein.
 27. The method according to claim 19 wherein saidDNA sequence encodes a sulphonylurea-tolerant AHAS.
 28. The methodaccording to claim 19 wherein said DNA sequence encodes aimdizalinone-tolerant AHAS.
 29. The method according to claim 19 whereinsaid DNA sequence encodes an enzyme of the carotenoid pathway.
 30. Themethod according to claim 29 wherein said DNA sequence is the crtI gene.31. The method according to claim 19 wherein said DNA sequence encodes abromoxynil-tolerant gene product.
 32. The method according to claim 31wherein said bromoxynil-tolerant gene is the bxn gene.
 33. A herbicidetolerant plant cell produced according to the method of claim
 19. 34. Aplant, plant seed or plant part each comprising a plant cell accordingto claim
 33. 35. A plant cell according to claim 33 and comprisinggreater than about 0.01% of total soluble protein as a protein expressedfrom said DNA sequence encoding a protein conferring tolerance to aherbicide wherein said ribosome binding site is from the bacteriophageT7 gene 10 leader.
 36. A plant cell according to claim 33 and comprisinggreater than about 0.1% of total soluble protein as a protein expressedfrom said DNA sequence encoding a protein conferring tolerance to aherbicide wherein said ribosome binding site is from the bacteriophageT7 gene 10 leader.
 37. A plant cell according to claim 33 and comprisinggreater than about 0.2% of total soluble protein as a protein expressedfrom said DNA sequence encoding a protein conferring tolerance to aherbicide wherein said ribosome binding site is from the bacteriophageT7 gene 10 leader.
 38. A plant cell according to claim 33 and comprising1% or more of total soluble protein as a protein expressed from said DNAsequence encoding a protein conferring tolerance to a herbicide whereinsaid ribosome binding site is from the bacteriophage T7 gene 10 leader.39. A plant cell according to claim 33 and comprising 12% or more oftotal soluble protein as a protein expressed from said DNA sequenceencoding a protein conferring tolerance to a herbicide wherein saidribosome binding site is from the bacteriophage T7 gene 10 leader.
 40. Aplant cell according to claim 33 wherein said DNA sequence encodes aglyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase.
 41. Aplant, plant seed or plant part each comprising a plant cell accordingto claim
 39. 42. A plant according to claim 41 tolerant of the herbicideglyphosate when said herbicide is applied at a rate of about 16 ouncesor greater per acre.
 43. A plant according to claim 41 tolerant of theherbicide glyphosate when said herbicide is applied at a rate of about32 ounces or greater per acre.
 44. A plant according to claim 41tolerant of the herbicide glyphosate when said herbicide is applied at arate of about 64 ounces or greater per acre.
 45. A plant according toclaim 41 tolerant of the herbicide glyphosate when said herbicide isapplied at a rate of about 128 ounces or greater per acre.
 46. Themethod according to claim 19, wherein said plant cells are tolerant toapplications of herbicide amounts selected from the group consisting of16 ounces/acre, 32 ounces/acre, 64 ounces/acre, and 128 ounces/acrewherein said ribosome binding site is from the bacteriophage T7 gene 10leader.
 47. The method according to claim 46, wherein said herbicide isglyphosate.